1) Background: Exposure of biologically active materials to ionizing radiation at very low temperature leads to damage to macromolecules. Biochemical functions are lost at a rate directly dependent on the molecular mass of the active structures. This is the basis for radiation target analysis which is used to determine the size of the functional unit of enzymes, receptors, transporters and others that carry out important biological activities. These studies revealed new and unexpected aspects of biomedically important systems including basic differences between proteins and ribonucleic acid in their responses to ionizing radiation. Other biophysical techniques are used to learn other aspects of macromolecular function in living cells: ultracentrifugation of intact Euglena cells results in stratification of cellular components based on their respective densities. These cells are living and can recover, grow and divide. 2) Objective of present studies: a: Fundamental studies of the actions of ionizing radiation on macromolecules continuing in order to define the exact nature of the damage in different species of molecules. Analyses of these effects by radiation target theory establishes a radiation-sensitive mass associated with measured biological activities. This reveals a fresh perspective in the structure-function relationship in these macromolecules. b: Application of the radiation technique to other enzymes, binding sites, and transporters to determine the size of their active structures, which often is less than the mass of the entire complex. c: Analysis of the recovery processes in stratified Euglena cells to determine the mechanisms involved and the time course of restoration of normal cellular function. 3) Results during the past year: a: Radiation studies of immunglobulin G (IgG). IgG structure involves two heavy chains of 52 kDa and two light chains of 24 kDa. In the rabbit, there are three disulfide bridges between these subunits: one at the terminus of each light chain linking to a heavy chain, and one weaker disulfide bridge between the two heavy chains. To test whether radiation damage can appear across these bridges, these disulfide bridges were chemically cleaved before radiation exposure. Radiation damage to each of the subunits was examined in both native and reduced irradiated samples. In both cases, radiation damage was condfined to a single subunit; there was no evidence for the transfer of radiation energy across a disulfide bridge. The 52 kDa subunits were destroyed individually whether or not they were linked by disulfide bridges to other subunits. The smaller subunits revealed a complex radiation response, indicating that small doses of radiation were less effective than high doses. Since only large and small subunits were present in the irradiated samples, it was concluded that radiation damage to the larger subunits released fragments which moved with the smaller subunits on electrophoresis. The mathematics of this model were found to agree quantitatively with the observations. These results raised many new questions which will be pursued. b: the recovery of stratified Euglena was found to depend on the g-force to which they had been exposed and the length of exposure. Centrifugal forces up to 150 000 x g and exposure times up to 18 hours were tested. The recovery process was due to a normal change in cell shape: contractile structures in the cell envelope caused the elongated cells to become more spherical; relaxation permitted the cells to become more extended. The internal contents of the cell became redistributed in this process. After one hour recovery time, the previously stratified cells were indistinguishable from untreated Euglena and metabolic activity was restored. 4) Conclusions and Significance: a) Radiation studies of IgG showed that molecular damage was restricted to the polypeptide which suffered a radiation interaction, even if the polypeptide was linked to other via a disulfide bridge. This result differs from previous reports of disulfide-linked ricin in which damage was observed in both chains only when the disulfide bridge was intact. While radiation damage appears throughout a polyptide no matter where the radiation event occurred, the mechanism(s) by which damage appears in adjacent polypeptides differs among proteins. b: The recovery of Euglena from centrifugation-induced stratification depends primarily on a natural process of changes in cell shape. The mechanism of this contractile operation is not yet defined, nor is the role of this process in normal cell metabolism.